Grizot Sylvestre, Salem Michèle, Vongsouthi Vanida, Durand Lionel, Moreau François, Dohi Hirofumi, Vincent Stéphane, Escaich Sonia, Ducruix Arnaud
Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS, Université Paris Descartes, Faculté de Pharmacie, 4, Avenue de l'Observatoire, F-75270 Paris cedex 06, France.
J Mol Biol. 2006 Oct 20;363(2):383-94. doi: 10.1016/j.jmb.2006.07.057. Epub 2006 Jul 29.
Lipopolysaccharides constitute the outer leaflet of the outer membrane of Gram-negative bacteria and are therefore essential for cell growth and viability. The heptosyltransferase WaaC is a glycosyltransferase (GT) involved in the synthesis of the inner core region of LPS. It catalyzes the addition of the first L-glycero-D-manno-heptose (heptose) molecule to one 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residue of the Kdo2-lipid A molecule. Heptose is an essential component of the LPS core domain; its absence results in a truncated lipopolysaccharide associated with the deep-rough phenotype causing a greater susceptibility to antibiotic and an attenuated virulence for pathogenic Gram-negative bacteria. Thus, WaaC represents a promising target in antibacterial drug design. Here, we report the structure of WaaC from the Escherichia coli pathogenic strain RS218 alone at 1.9 A resolution, and in complex with either ADP or the non-cleavable analog ADP-2-deoxy-2-fluoro-heptose of the sugar donor at 2.4 A resolution. WaaC adopts the GT-B fold in two domains, characteristic of one glycosyltransferase structural superfamily. The comparison of the three different structures shows that WaaC does not undergo a domain rotation, characteristic of the GT-B family, upon substrate binding, but allows the substrate analog and the reaction product to adopt remarkably distinct conformations inside the active site. In addition, both binary complexes offer a close view of the donor subsite and, together with results from site-directed mutagenesis studies, provide evidence for a model of the catalytic mechanism.
脂多糖构成革兰氏阴性菌外膜的外层小叶，因此对细胞生长和活力至关重要。庚糖基转移酶WaaC是一种糖基转移酶（GT），参与脂多糖内核区域的合成。它催化将第一个L-甘油-D-甘露庚糖（庚糖）分子添加到Kdo2-脂质A分子的一个3-脱氧-D-甘露辛-2-酮糖酸（Kdo）残基上。庚糖是脂多糖核心结构域的重要组成部分；其缺失会导致与深粗糙表型相关的截短脂多糖，使革兰氏阴性病原菌对抗生素更敏感且毒力减弱。因此，WaaC是抗菌药物设计中一个有前景的靶点。在此，我们报告了来自大肠杆菌致病菌株RS218的WaaC单独的结构，分辨率为1.9 Å，以及与ADP或糖供体的不可裂解类似物ADP-2-脱氧-2-氟庚糖形成复合物的结构，分辨率为2.4 Å。WaaC在两个结构域中采用GT-B折叠，这是一个糖基转移酶结构超家族的特征。对这三种不同结构的比较表明，WaaC在底物结合时不会发生GT-B家族特有的结构域旋转，但允许底物类似物和反应产物在活性位点内采取明显不同的构象。此外，这两种二元复合物都提供了对供体亚位点的近距离观察，并与定点诱变研究的结果一起，为催化机制模型提供了证据。
